【摘要】：背景腎功能不全(renalinsufficiency)是各種復雜因素導致腎小球損害,腎功能降低,造成人體水電解質(zhì)酸堿平衡失常,代謝紊亂的臨床綜合癥候群。腎功能不全是一種發(fā)病率高,危害較大,嚴重危害患者健康與生命的病癥之一。目前腎功能不全的發(fā)生進展機制都還不夠完善。我們希望通過血清蛋白質(zhì)組學研究發(fā)現(xiàn)腎功能不全大鼠與正常大鼠的差異蛋白,為腎功能不全的進展研究提供實驗基礎(chǔ)。目的運用iTRAQ技術(shù)進行蛋白質(zhì)組學分析,研究大鼠腎功能不全發(fā)生進展的相關(guān)差異蛋白;運用omicsbean在線軟件,通過GO分析、KEGG分析和STRING等生物信息學分析對篩選出的差異蛋白進行分析,并對與腎功能不全相關(guān)的蛋白質(zhì)進行鑒定及驗證。方法把44只SD雄性大鼠隨機分為正常對照組(A組),模型低劑量組(B組),模型中劑量組(C組)和模型高劑量組(D組),各11只,正常對照組常規(guī)飼養(yǎng),模型輕劑量組,模型中劑量組和模型高劑量組大鼠分別給予20g/d/只含0.125%、0.25%、0.5%腺嘌呤食物,連續(xù)21d。造模結(jié)束后,測驗各組大鼠BUN(尿素氮)、Scr(血肌酐),體質(zhì)量,24h飲水量,HE染色檢測腎組織形態(tài)學改變。利用iTRAQ技術(shù)分析血清蛋白質(zhì)組,鑒定出差異表達蛋白,進一步用實時定量PCR、蛋白質(zhì)印跡及酶聯(lián)免疫吸附實驗等實驗對觸珠蛋白(Haptoglobin,HP),正五聚蛋白-3(Pentaxin,PTX3),血清轉(zhuǎn)鐵蛋白(Serotransferrin,TRF)的表達水平加以驗證。結(jié)果通過對各模型組大鼠與正常對照大鼠血清的質(zhì)譜分析,我們共發(fā)現(xiàn)416種蛋白質(zhì),并從中成功鑒定出差異表達蛋白29種。利用omicsbean生物信息學在線軟件分析,發(fā)現(xiàn)這29種差異蛋白主要分布在細胞外區(qū)域,血液微粒和膜界囊泡中,以分子功能調(diào)節(jié)和酶調(diào)節(jié)活性等功能為主,參與炎癥反應(yīng)以及對外部刺激應(yīng)答等生物學過程。通過查閱文獻和結(jié)果分析,從29種差異表達蛋白中篩選了3種蛋白,即HP、PTX3和TRF。蛋白質(zhì)印跡、實時定量PCR和酶聯(lián)免疫吸附實驗結(jié)果與質(zhì)譜結(jié)果一致。結(jié)論成功建立了腎功能不全大鼠模型,并基于iTRAQ技術(shù)篩選出了各期腎功能不全大鼠與正常大鼠的差異蛋白,經(jīng)生物信息學分析,腎功能不全的發(fā)生與多個蛋白質(zhì)、生物學進程、蛋白通路及蛋白質(zhì)調(diào)控網(wǎng)絡(luò)有關(guān),提示HP、PTX3和TRF可能與腎功能不全的發(fā)生進展密切相關(guān),為腎功能不全的藥物靶點治療提供了一定實驗基礎(chǔ)。
[Abstract]:Background (renalinsufficiency) of renal insufficiency is a clinical syndrome with various complex factors leading to glomerular damage and decrease of renal function, resulting in abnormal acid-base balance and metabolic disorder of hydrolytic acid and base in human body. Renal insufficiency is one of the diseases with high incidence, great harm and serious harm to the health and life of patients. At present, the mechanism of the occurrence and progress of renal insufficiency is not perfect. We hope that the differential proteins between rats with renal insufficiency and normal rats will be found by serum proteome study, which will provide an experimental basis for the progress of renal insufficiency. Objective to study the differential proteins related to the progress of renal insufficiency in rats by proteome analysis with iTRAQ technique. Omicsbean online software, GO analysis, KEGG analysis and STRING bioinformatics analysis were used to analyze the screened differential proteins, and the proteins related to renal insufficiency were identified and verified. Methods 44 male SD rats were randomly divided into normal control group (group A), model low dose group (group B), middle dose group (group C) and model high dose group (group D) with 11 rats in each group. Rats in the model light dose group, the middle dose group and the model high dose group were given 20g/d/ containing only 0.125%, 0.25% and 0.5% adenine food for 21 days. After the establishment of the model, BUN (urea nitrogen), Scr (serum creatinine), body mass, 24 h drinking water and HE staining were used to detect the morphological changes of renal tissue. The serum proteome was analyzed by iTRAQ, and the differentially expressed proteins were identified. Haptoglobin,HP and Pentaxin,PTX3 were further detected by real-time quantitative PCR, Western imprinting and enzyme-linked immunosorbent assay (Elisa). The expression level of serum transferrin (Serotransferrin,TRF) was verified. Results by mass spectrometry analysis of serum of rats in each model group and normal control group, 416 kinds of proteins were found, and 29 kinds of differentially expressed proteins were successfully identified. Using omicsbean bioinformatics online software analysis, it was found that the 29 differential proteins were mainly distributed in extracellular region, blood particles and membrane vesicles, mainly in molecular function regulation and enzyme regulation activity. Participate in biological processes such as inflammatory response and response to external stimuli. Through literature review and result analysis, three proteins, HP,PTX3 and TRF., were screened from 29 differentially expressed proteins. The results of Western imprinting, real-time quantitative PCR and enzyme-linked immunosorbent assay were consistent with those of mass spectrometry. Conclusion the rat model of renal insufficiency was successfully established, and the differential proteins between rats with renal insufficiency and normal rats were screened by iTRAQ technique. The occurrence of renal insufficiency and the occurrence of multiple proteins and biological processes were analyzed by bioinformatics analysis. Protein pathway and protein regulatory network are related, suggesting that HP,PTX3 and TRF may be closely related to the occurrence and progress of renal insufficiency, which provides an experimental basis for drug target therapy of renal insufficiency.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692

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